Chimeric antigen receptor-based natural killer cell immunotherapy in cancer: from bench to bedside

Immunotherapy has rapidly evolved in the past decades in the battle against cancer. Chimeric antigen receptor (CAR)-engineered T cells have demonstrated significant success in certain hematologic malignancies, although they still face certain limitations, including high costs and toxic effects. Natural killer cells (NK cells), as a vital component of the immune system, serve as the “first responders” in the context of cancer development. In this literature review, we provide an updated understanding of NK cell development, functions, and their applications in disease therapy. Furthermore, we explore the rationale for utilizing engineered NK cell therapies, such as CAR-NK cells, and discuss the differences between CAR-T and CAR-NK cells. We also provide insights into the key elements and strategies involved in CAR design for engineered NK cells. In addition, we highlight the challenges currently encountered and discuss the future directions in NK cell research and utilization, including pre-clinical investigations and ongoing clinical trials. Based on the outstanding antitumor potential of NK cells, it is highly likely that they will lead to groundbreaking advancements in cancer treatment in the future.


INTRODUCTION
Cancer, a significant class of diseases, ranks among the leading causes of human mortality.This malignant disease now stands as the primary cause of death, second only to cardiovascular and cerebrovascular diseases [1].In addition to traditional treatment methods such as surgery, chemotherapy, and radiotherapy [2], current cancer therapies have rapidly evolved, including targeted therapy and immunotherapy [3].Targeted therapy is based on the advancements of cellular and molecular biology, and it seeks to inhibit cancer cells by identifying specific target molecules expressed by these cells.While notable progress has been made in enhancing therapeutic effects and reducing side effects, challenges such as drug resistance and off-target effects continue 1 Institute of Biomedical Research, Yunnan University, Kunming 650500, China. 2 Graduate School of Capital Medical University, Beijing 100069, China. 3Department of Oncology, Wuming Hospital of Guangxi Medical University, Nanning 530199, China. 4Department of Hematology, Huazhong University of Science and Technology Union Shenzhen Hospital, Shenzhen 518052, China. 5Department of Gastroenterology, The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530000, China.
to persist in targeted therapies.On the other hand, immunotherapy has rapidly developed in recent years.It is primarily divided into two categories: immune checkpoint blockade therapy [4] and immune cell therapy [5].These approaches have yielded promising clinical recoveries for certain patients, indicating a bright future for cancer treatment.
Natural killer (NK) cells, as a vital type of immune cell, possess dual functions involving cytotoxicity and immune regulation [6].These cells originate from hematopoietic stem cells (HSCs) in the bone marrow and acquire the ability to differentiate between "friend" and "foe" through self-major histocompatibility complex (MHC) I molecule recognition [7].NK cells exert broad-spectrum anticancer effects without the need for specificity and MHC restriction typically observed in cancer cell inhibition.They act as the "first responders" in the battle of the immune system against cancer development and viral infections.NK cells can spontaneously identify aberrant cells in the body, swiftly eliminating them via cytotoxic means while simultaneously generating various pro-inflammatory cytokines and chemokines.Furthermore, they have the capacity to activate other immune cells, initiating the adaptive immune response [8].NK cells were initially discovered in the 1970s and primarily play a role in combating infected microorganisms and malignantly transformed allogeneic and autologous cells.As a distinct population of innate lymphocytes, NK cells inherently possess the ability to recognize and eliminate virus-infected and cancer cells.This recognition and elimination capacity rendered NK cells safe and effective as immunotherapeutic agents for patients with advanced leukemia nearly two decades ago [9].In recent years, NK cells have received increasing attention as potential candidates for immunotherapy.
With the rapid development of immunotherapy, especially in the realm of cell therapy, research on NK cells has become increasingly intensive due to their pivotal role in disease treatment.In this paper, we aim to elucidate the biological background, cell sources, and the use of NK cells in disease therapy.We have also discussed the principal advantages and disadvantages of engineered NK cells, offering valuable insights into future avenues for NK cell therapy research.

NK CELL BIOLOGY NK cell origination, development and classification
Peripheral blood mononuclear cells (PBMCs) stem from HSCs in the bone marrow, responsible for generating all the blood cells in the immune system through hematopoiesis [10].HSCs possess the capacity to differentiate into two primary lineages: myeloid (including monocytes, macrophages, granulocytes, megakaryocytes, dendritic cells, and erythrocytes) and lymphoid (involving T cells, B cells, and NK cells).In healthy human bodies, approximately 70-90% of PBMCs are lymphocytes, and the majority of T cells remain in a naive state (mature but not stimulated by antigens).Only a small fraction of T cells become activated upon antigen recognition, initiating a cell-mediated immune response.Similarly, in normal human bodies, B cells predominantly exist in a naive or memory state, awaiting antigen stimulation and constituting approximately 5-15% of lymphocytes.Upon activation, B cells differentiate into plasma cells, which release substantial quantities of antibodies, triggering a humoral immune response.NK cells account for approximately 5-10% of the lymphocyte population and play a crucial role in the body's innate immunity [11].
The development of NK cells primarily occurs from common lymphoid progenitors (CLPs) in the bone marrow, progressing through distinct stages, mainly including NK precursors (NKP), immature NK cells, and mature NK cells as illustrated in Fig. 1.In the course of normal hematopoiesis, one of the earliest markers of NK cells is IL2RB (CD122), which is expressed when CD34 + CLPs enter the NK cell fate lineage.Recent research has revealed the complexity of NK cell development.A "branched" model associated with NK cell development has been described, and the investigation into NK cell trafficking, tissue residence, and tissue-specific specialization is ongoing [12][13][14][15][16]. Subpopulations of NK cell precursors can be differentiated by their varied gene expression, including markers such as CD34, KIT, KLRB1, CD244, and IL-15R [17].To identify immature NK cells, molecular markers like CD244, natural toxicity receptor (NCR)1, NCR2, NCR3, and KLRB1 have been reported.Subsequent differentiation to the mature stage is characterized by the expression of markers such as KLRD1, ITGB2, killer Ig-like receptors (KIRs), PRF1, IFNG, CD56, and CD16 [18].The CD56 bright subset is often considered an early stage of NK cell maturation [19].Notably, CD56 bright cells exhibit a higher proliferative capacity than CD56 dim cells, likely due to their prominent expression of several key proteins, including CCR7, CSF2, CXCR3, IL2RB, KLRC1, and SELL [20].This subpopulation is known for its elevated IFNG expression.Downregulation of CD56 is followed by CD16 expression, resulting in the CD56 dim NK cell subset [21].This subgroup is distinguished by its potent cytotoxic activity [22,23], with KIRs being among the genes associated with this subgroup [24].Additional genes linked to the CD56 dim subset include CX3CR1 [25], KLGR1 [26], and PRF1 [12].
While the CD56 bright cells make up a smaller portion, estimated at only 5-10% of the total NK cell population in peripheral blood (PB), the CD56 dim NK cells constitute over 90%.However, CD56 bright cells are more prevalent in specific tissues, including secondary lymphoid tissues [11,21].Recent findings have suggested that NK cell development can also take place in secondary lymphoid tissues such as lymph nodes and the spleen [27].During this developmental phase, NK cells are trained to recognize MHC-I, thereby avoiding the targeting of healthy, normal cells.
NK cell membrane protein characteristics and biology functions NK cells eliminate infected and cancerous cells upon activation of activating receptors, such as the NCRs, which include NKp30, NKp44, and NKp46 [28][29][30].As mentioned earlier, CD56 dim NK cells primarily display robust cytotoxic activity, whereas CD56 bright NK cells primarily secrete cytokines and exhibit lower cytotoxic activity.The regulation of NK cell cytotoxicity involves a complex interplay of activation and inhibition signals.The killing efficiency Fig. 1 The maturation steps of NK cells in hematopoietic stem cell (HSC) differentiation and hematopoiesis process.
of NK cells on target cells relies on the delicate balance between inhibitory and activation signals, closely tied to cell membrane receptors and proteins (Fig. 2).Activated NK cells employ various mechanisms for target cell elimination (Fig. 3), including ① release of perforin and granzyme, leading to target cell lysis or apoptosis; ② promotion of FasL expression, inducing cell apoptosis; ③ release of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokines (such as CCL1, CCL2, CCL3, CCL4, CCL5, and CXCL8) to recruit and activate other effector immune cells; ④ antibody-dependent cellmediated cytotoxicity (ADCC).NK cells express various membrane proteins, as depicted in Fig. 2, to modulate inhibitory and activation signals [16]; however, they lack T-cell receptors (TCRs) and B-cell receptors, among which CD56 [31] (an adhesion molecule mediating homotypic adhesion) and CD16 [32] (a lowaffinity Fc receptor FcγRIII, contributing to NK cell-mediated ADCC) serve as primary surface markers.Unlike T cells, NK cells do not express antigen-specific recognition receptors.The surface receptors of NK cells are broadly categorized into activating and inhibitory receptors, both capable of recognizing classical or nonclassical human MHC-class I molecules on normal cells.The interaction between MHC-I molecules and inhibitory receptors signals convey a "Do not eat me" message to NK cells, enabling cell evasion from NK cell-mediated killing [33].

NK CELL ENGINEERING
Numerous studies suggest the presence of potential neoantigens, such as HSP70, expressed exclusively on tumor cells and absent in normal cells, can activate NK cells.This discovery opens a promising avenue in target-driven cell-based immunotherapies, as the HSP70 protein becomes a viable target for NK cells [34][35][36].Currently, several approaches exist in NK cell engineering (Fig. 4).A prominent method involves chimeric antigen receptors (CARs), receptor proteins that confer immune cells with the ability to target specific antigenic proteins.CAR-T cell therapy has achieved significant success in hematological malignancies, including acute lymphoblastic leukemia [37,38] and diffuse large B-cell lymphoma [39].Various CAR-T therapies have gained approval [40][41][42].While CAR-T cell therapy stands as a groundbreaking biotechnological advancement in cancer treatment, it faces challenges such as side effects, toxicity [43], T cell exhaustion [44], and limited efficacy against solid tumors [45].Presently, new cell therapies such as CAR-NK, CAR-NKT, CAR-macrophage (CAR-M), CAR-Treg, CAR-γδT, with CAR technology at their core, have emerged, especially CAR-NK showing promising prospects in tumor immunotherapy [46].

NK cell sources
There are four primary sources of NK cells in NK cell engineering (e.g., CAR-NK), depicted in Fig. 5: Peripheral blood (PB), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (e.g., umbilical cord blood), and NK cell lines (such as NK-92, etc.).As previously mentioned, NK cells, as a subset of lymphocytes, possess the ability to recognize and eliminate tumor cells without prior sensitization, antibody involvement, and are not restricted by MHC.Commonly utilized markers for NK cells in experiments include CD16 and CD56.The characteristics and limitations of NK cells from different sources in clinical treatment are detailed below.
Peripheral blood (PB-NK).NK cells derived from the patient's own body or a healthy donor.Due to disease and treatment limitations, the function of the patient's own NK cells may be compromised.Allogeneic NK cells are clinically favored [47], but careful T cell removal is essential to mitigate graft-versus-host disease (GVHD) [48].PB-NK cells are mature and do not necessitate induction of differentiation, but gene transduction efficiency is relatively low.Prolonged in vitro expansion may result in shortened telomeres and reduced cytotoxicity.Cryopreservation diminishes the viability and toxicity of PB-NK cells.NK cell therapy typically involves    4 Approaches in NK cell engineering against cancer.1) CAR-NK cells: NK cells that are modified to express extracellular receptors (such as scFvs, nanobodies, peptides, and ligands) for HLA-independent antigen recognition.2) TCR NK cells: NK cells that are modified to express TCR complexes for MHC-I presented cancer antigens.3) Multi-specific Ab-based NK cell therapy: NK cells that utilize mono-antibodies, bispecific antibodies, or tri-specific antibodies to recognize cancer antigens (including but not limited to bi-specific NK-cell-engager, BiKE).reinfusion of 10 6 -10 8 cells per kilogram of body weight [49,50], yet the proportion of NK cells in PB is low [51], posing challenges for large-scale in vitro culture.However, there are advantages; for instance, these cells are mature and bypass the need for an extended differentiation period.
Umbilical cord blood (UCB-NK).These cells exhibit high proliferation efficiency, allowing real-time selection of human leukocyte antigen (HLA)-mismatched products by establishing an NK cell bank [52].Cord blood serves as a promising source for NK cells in clinical applications, with two main strategies: direct use of NK cells in cord blood or differentiation of HSCs in cord blood into NK cells.It is crucial to note that mesenchymal cells in cord blood are not utilized for these purposes.Cord blood remains a relatively stable source of NK cells.Due to their high proliferative capacity, only 10% of a cord blood unit is required to generate a nearly pure cell pool of over 10 9 NK cells within 2 weeks, typically suitable for one treatment cycle [53,54].However, UCB-NK cells are not fully differentiated, exhibiting relatively low expression of NK receptors and limited cell inhibition ability, with a potential risk of tumorigenesis in allogeneic transplantation [55].Nevertheless, they demonstrate a robust bone marrow homing ability [56].Moreover, the higher proportion of hemoglobin and red blood cells in cord blood can impact the isolation and culture of PBMCs.
Stem cell-derived NK cells.NK cells are commonly induced from human embryonic stem cells (hESCs), HSCs or iPSCs [57], with an expansion period extending beyond 3-5 weeks.This extended period helps mitigate the heterogeneity of NK cells between the recipient and the donor.However, NK cells induced by iPSCs present potential risks of malignant transformation and tumorigenesis in vivo, along with the possibility of triggering unexpected immune responses due to their potential immunogenicity.iPSCs efficiently clone, expand, and differentiate in vitro, producing a substantial quantity of uniform NK cell products [58].Nevertheless, iPSC-derived NK cells often express low levels of endogenous CD16, a drawback that can be addressed through genetic engineering [59,60].Moreover, iPSCs may retain DNA methylation signatures consistent with their somatic tissue origin, contributing to "epigenetic memory," which could influence the development of specific cell lineages distinct from the donor cells.Systemic administration of cytokines in a clinical setting is highly undesirable due to its expense and potential dangers.Additionally, iPSC-derived cells carry the risk of malignant transformation and potential immunogenicity, leading to the destruction of ES cells and even adverse immune responses such as cytokine release storms [61].
NK cell line.Various NK cell lines, including NK-92, HANK-1, KHYG-1, NK-YS, NKG, NK101, NK3.3, YTS, and NKL, have been constructed to date, serving as excellent cell models for studying NK cell biology and associated applications.Among these, the NK-92 cell line is the only one applied in clinical trials, demonstrating a relatively satisfactory response outcome with controllable adverse effects.NK-92 cells lack the CD16 receptor-mediated ADCC effect, but this can be addressed through modification [62].These cells are easily genetically manipulated, allowing effective introduction of exogenous genes through electroporation without the need for viral vectors [63].Because NK-92 is a tumor-derived aneuploid immortalized cell line [64], it requires irradiation before use to inhibit in vivo proliferation [65], negatively impacting long-term persistence and overall therapeutic potential.

CAR-NK cells
CAR-NK cells typically share the similar CAR structures as CAR-T cells.NK cells enhance their cytotoxic capacity and cytokine production through co-stimulatory molecules like NKG2D and CD244, providing probably stronger tumor-specific targeting and cytotoxicity than CAR-T cells [66].CAR-NK cell therapy is a potential alternative to CAR-T therapy due to several unique features.Firstly, allogeneic NK cells are generally safe for adoptive cell therapy (ACT) as they do not typically mediate GVHD [66].Moreover, NK cells only secrete small amounts of IFN-γ and GM-CSF [67], without producing IL-1 and IL-6 that initiate cytokine release syndrome (CRS) [68].Secondly, in addition to inhibiting cancer cells through single-chain antibody recognition of tumor surface antigens, NK cells can also recognize various ligands through multiple receptors such as NCRs (NKp46, NKp44, and NKp30), NKG2D, and DNAM-1 (CD226) [69][70][71].Lastly, NK cells are abundant in clinical samples [13] and can be generated from various sources, including PB [72], UCB [54], hESCs, iPSCs [57], and even NK-92 cell lines [73] as mentioned above.
Similar to CAR-T cells, the functional CAR molecule expressed on NK cells comprises three components: an extracellular domain, a transmembrane region, and an intracellular signaling domain (Fig. 6A).The extracellular domain includes a signal peptide and a single-chain antibody fragment (scFv) responsible for recognizing the antigen.A hinge region connects this structure to the transmembrane region, which, in turn, links to the intracellular domain containing the activation signal.The commonly utilized transmembrane segment for CAR-NK is adapted from CD3ζ, CD8, or CD28, with T cell-specific CD8 and CD28 being the most frequently employed [74].The intracellular segment is pivotal for cell activation post-reception of the target antigen signal and constitutes a linear structure of co-stimulatory molecules and signaling domains recruited downstream of signal transduction.Successful CAR design is achieved via a combination of meticulous design and functional testing.The evolution of CAR generations includes the first generation containing only CD3ζ [75], the second and third generations adding one or two co-stimulatory domains, respectively, based on the first generation [76], the fourth generation incorporating a cytokine secretion segment based on the third generation [77], and the fifth generation introducing a special binding motif (Fig. 6A).Logic-gated control of CAR cells has also been developed to achieve precision therapy and avert potential toxicity (Fig. 6B).Currently, the second-generation CAR structures CD28-CD3ζ and 41BB-CD3ζ are most commonly used in the field of CAR-NK, whereas in the third generation, CD28-41BB-CD3ζ is also frequently employed [74,78].A detailed description of each CAR element is discussed below.
Vector backbone and promoter.The vector backbone incorporates all elements necessary for CAR expression, including a promoter, polyA signal, and transcriptional regulatory fragments.The choice of promoter directly impacts the expression of the transgene.Current reports on CAR-NK cells reveal the use of various promoters to drive CAR expression, whether derived from cell lines [79] or primary NK cells [80].In primary CAR-NK and CAR-NK cell lines, viral promoters (such as MPSV and MMLV) are more commonly utilized than constitutively active promoters (such as EF1α and PGK) [81].
Signal peptide.Signal peptides exhibit substantial heterogeneity, leading to varying levels of protein production efficiency.For CAR-NK and CAR-T cells, comparative studies identifying the optimal signal peptide are lacking.Currently, CD8a-SP is the most commonly used signal peptide sequence for NK cells, and immunoglobulin heavy or light chain signal peptides are reported for NK cell lines [82].
Single-chain antibody fragment (scFv).The scFv serves as the tumor antigen-binding domain of CAR [83], determining the specificity and function of CAR-NK cells.As single-chain antibodies deviate from the natural form of antibodies, the order of the heavy and light chains is artificially determined [84].For CAR-NK designs, the VH-VL direction is preferred over the VL-VH direction [85][86][87].Fujiwara et al. revealed that the order of the heavy and light chains does not affect the expression of CARs on T cells [88].
Furthermore, cells can be equipped with multiple scFvs, thereby expanding the antigen recognition capacity of CAR effector cells.Several options exist: the CAR can be transduced with a twoelement vector, inducing the expression of two CAR constructs; or two scFvs can be fused in one construct, creating a "single-handle" CAR with tandem scFvs [44,89].While these technologies have been utilized to produce CAR-T cells [90], their application in CAR-NK cells is not well-documented.In most current clinical CAR-T cell trials, single-chain antibodies derived from mouse antibodies are commonly used, increasing the risk of GVHD in anti-mouse IgG cells.This risk can be mitigated through humanization or screening of fully human antibodies [91].Unfortunately, even humanized scFvs may induce host anti-idiotypic immune responses due to the chimeric nature of CAR receptors [92].However, in the limited number of CAR-NK clinical trials to date, no major adverse effects associated with anti-CAR immune responses have been identified [66].Moreover, several other forms like nanobody were also explored to serve as the tumor antigen-binding domain.
Linking region.The linking region between the heavy and light chains contributes to stabilizing the conformation of the singlechain antibody.A too-short linking region may lead to multimer formation, whereas a too-long linking region can cause hydrolysis or reduce the association between VH and VL domains [93].For CAR-NK cells, the GGGGS pentapeptide is widely used in multimers, typically in 3 repeats.Another linker designed to enhance proteolytic stability is the Whitlow "218" linker (GSTSGSGKPGSGEGSTKG) [88,94].
Hinge region.The hinge region, the extracellular domain of the CAR connecting the single-chain antibody unit and transmembrane domain, maintains the stability required for robust CAR expression and activity in effector cells.Most CAR-NK constructions use derivatives of CD8α or CD28 extracellular domains or IgG-based hinge regions.The type and length of the hinge region significantly affect the functional activity of CAR [95].Although most information comes from CAR-T, the direct transformation into CAR-NK remains unproven.A direct comparison between CD28 and CD8α hinge regions revealed that CD28 is more likely to promote CAR molecule dimerization, resulting in a stronger activation stimulus [93].While beneficial, this can also lead to more serious adverse effects.IgG-based hinge regions, made up of the Fc portion of IgG1 or the CH2/CH3 domains of the Fc portion, offer flexibility in structure.The length of the hinge region can be adjusted to adapt to antigen recognition; however studies have revealed that a shorter spacer region results in higher cytokine production, faster CAR cell proliferation, and improved persistence and antitumor effects in vivo [95].
Transmembrane domain.The transmembrane domain connects the CAR extracellular domain and the intracellular activation signal domain.The most commonly used transmembrane domains for CAR-NK originate from CD3ζ, CD8, and CD28.The choice of transmembrane domain influences the activation extent of the CAR construct in cellular functions.Transmembrane domains from molecules typically expressed on NK cells, such as DNAM-1, 2B4, and NKG2D, lead to increased CD107a degranulation and higher cytotoxicity.Thus, the specific source of the transmembrane domain determines the activity of CAR-NK [96].An important aspect of the transmembrane domain is that it should follow the natural orientation (order of N-terminal to C-terminal) of transmembrane proteins on NK cells.At present, CD8α-and CD28-modified transmembrane regions are most common in primary CAR-NK cells, whereas CD28 is the preferred transmembrane region for CAR-NK cell lines [93].
Activation signal.The number of intracellular activation signals in a CAR determines its "generation".First-generation CAR-NK cells, akin to CAR-T cells, contain only the CD3ζ signal.Secondgeneration and third-generation CAR-NKs carry one and two additional co-stimulatory signals, respectively, typically derived from the CD28 family (CD28 and ICOS), the TNF receptor family (4-1BB, OX40, and CD27), or the signaling lymphocytic activation molecule-related receptor family (2B4) [97].The published CAR-NK clinical trial used a second-generation CAR-NK construct that improved activity by incorporating IL-15 expression.Most current CAR structures depend on the CD3ζ chain signaling domain, and robust activation signals are crucial for eliciting potent antitumor responses but may also result in rapid effector cell exhaustion.Combinations of co-stimulatory domains can be employed to calibrate desired immune cell responses.CD28-based CARs exhibit a faster effector profile than 4-1BB-based CARs, inducing higher levels of IFN-γ, granzyme B, and TNF-α.However, this strong costimulatory signal also results in activation-induced cell death (AICD).Conversely, 4-1BB-CD3ζ signaling preferentially induces memory-related genes and sustained antitumor activity [98,99].This difference may be attributed to the amelioration of T cell exhaustion induced by the 4-1BB domain in contrast to the CD28 domain [44].CD3ζ was universally used as the primary activation domain in studies of CAR-NK cell lines and primary CAR-NK cells, with approximately half carrying an additional activation domain, generally with the addition of 4-1BB or CD28.For third-generation constructs, the combination CD28/4-1BB/CD3ζ is most commonly employed.Intracellular signaling domains, such as CD28, 4-1BB, and OX40, often function to trigger immune cell activation and inhibition [100].A recent report using iPSC-derived CAR-NK therapy identified the crucial role of the NKG2D transmembrane domain and emphasized the critical role of the 2B4 co-stimulation domain [96].Different researchers also performed similar studies using 2B4 to highlight the importance of the activation signal in immune cell therapy [101,102].At present, four generations of CAR structures have been developed and are available for CAR-NK research.
CAR transfection or transduction vector.With the advancement of gene modification technology, various methods have been employed to generate CAR-NK cells.The two primary methods include viral transduction [103] (using lentiviruses or retroviruses) and transfection of naked plasmid DNA [87], transposase DNAmediated integration [104], and mRNA electroporation [96].Lentiviruses can efficiently transduce both periodic and non-cyclical cells and have been widely utilized in gene therapy [105].They have been successfully used as vectors in studies on primary CAR-NK cells and CAR-NK cell lines.Both secondgeneration and third-generation lentiviruses have been used in preclinical studies to generate CAR-expressing NK cell lines and primary CAR-NK cells.Retroviral vectors are also commonly used for NK cells [54,66].Retroviruses have been used as gene therapy vectors for decades, including CAR-NK cell lines and primary NK cells [106].In a recent phase I clinical trial, retroviral-transduced CD19 CAR-NK cells were used to treat CD19 + non-Hodgkin's lymphoma and chronic lymphocytic leukemia.The study results stated that 73% of patients responded to the treatment, with 7 of 8 patients achieving a complete response.Responses were rapid, occurring within 30 days of CAR-NK administration at all dose levels.After the 1-year follow-up, expanded CAR-NK cells remained detectable [66].Following infusion, CAR-NK DNA copy numbers remained stable in PB for up to 1 year, indicating, for the first time, that retroviral-transduced CAR-NK cells can exhibit longterm in vivo survival.Different retroviruses types have been used to generate CAR-NK cells.The RD114 retrovirus was reported to be more efficient at transducing primary NK cells than the γ retrovirus and lentivirus [107].Although long-term stable CAR expression in NK cells can be achieved using various retroviruses, the safety of retroviral systems remains a concern, especially when compared with the safer lentiviruses.CAR-encoding mRNA electroporation is a rapid, efficient, but short-lived method.To date, mRNA electroporation has been used in CAR-NK cell lines and primary CAR-NK cell studies.Generally, expanded or activated NK cells exhibit much higher mRNA transfection efficiency than freshly isolated NK cells [108].Because mRNA synthesis is a good manufacturing practice (GMP)-compliant manner and electroporation can be conducted in a clean room, it is feasible to generate GMP-compliant CAR-NK cells through mRNA electroporation.However, the primary disadvantage of this approach is the short window for CAR expression: after electroporation, CAR-NK cells should be infused back into the patient within 7 days.The Sleeping Beauty transposon system has also been developed.Transposon-based systems offer important advantages over conventional methods, such as the efficient introduction of CAR transgenes at predetermined locations.Transposons are primarily introduced into NK cells via electroporation and then integrated into the host genome by transposonases [50].Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is another powerful genetic modification technology that introduces the Cas9 protein into NK cells along with gRNA.This technique was initially used to disrupt the CD38 gene in primary NK cells [109], aiming to prevent NK cell cannibalism in combination with daratumumab (anti-CD38) because CD38 is expressed in NK cells, multiple myeloma [110], and acute myeloid leukemia (AML) cells [111].CRISPR/Cas9 has also been recently used to introduce some other new genes [112].Overall, using CRISPR/Cas9 strategy is a promising strategy to precisely delete, repair, or introduce specific genes, facilitating the generation of potent antitumor NK cells.
While we have established the superiority of a specific domain among multiple candidates based on findings from T cells expressing the CAR applied into NK cells, it is crucial to note that the superiority of CAR domains is complex and depends on factors such as the target and the interaction with other domains.Further exploration is needed to determine if the results and experiences obtained from T cells are applicable to NK cells and to anticipate optimization.Moreover, the expansion of NK cells may result in an adverse phenomenon of "suicide" or "fratricide" as mentioned above, where cells recognize receptors or ligands on the surface of other similar cells and trigger cytotoxic activity against them.The Fas/FasL axis is among the most relevant mechanisms.FasL-mediated cytotoxicity plays a crucial role in NK cell function, triggering caspase-dependent apoptosis when it binds to the receptor Fas in the target cell.Fas can also serve as a steady-state mechanism for inhibiting NK cell activity expressed by NK cells, known as AICD.NKG2D is another receptor that may lead to self-killing among NK cells, which is a natural receptor primarily comprising NK, CD8 + T, and γδ T cell expression, displaying recognition of various stress-induced ligands.Cannibalism may also occur in CAR-NK cells due to CAR ligand/antigen recognition if certain target antigens are also expressed on these effector cells [113].Furthermore, trogocytosis is a common phenomenon that often takes place during NK cell-mediated cancer inhibition.Trogocytosis ultimately leads to the transfer of antigens to NK cells, mediating the inhibition of NK cells by other NK cells.The knock out of target antigens in effector cells can overcome fratricide, but this method is unsuitable for antigens transferred to effector cells during trogocytosis.Low affinity of CAR to antigen or optimized CAR signaling transduction may serve as alternative approaches [114].

Popular targets of CAR-NK research
Solid tumor targets.In several cancer types, programmed cell death ligand 1 (PD-L1) is upregulated in the tumor microenvironment (TME) and in immunosuppressive cells [115].Preclinical tests have revealed that PD-L1-targeted CAR-NK cells exhibit specific antitumor effects against several in vitro tumor cell lines, and exhibit robust in vivo antitumor effects against triple-negative breast cancer [116], bladder cancer [117], and lung cancer [118].Human epidermal growth factor receptor 2 (HER2)/erythroblastic oncogene B 2 (ERBB2) is often overexpressed in breast, gastric, esophageal, ovarian, and endometrial cancers [119].HER2 is associated with poor survival and also expressed in most glioblastomas [120].Extensive studies have been conducted on the application of CAR constructs targeting HER2 [121].NKG2D is an activating NK cell receptor modulating the anticancer cytotoxic potential of NK cells by interacting with its tumor-associated overexpressed ligands [122].NKG2D ligands include MICA, MICB, and ULBPs (ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6) [123,124].Altogether, the targets in solid tumor (pre-) clinical therapy are relatively fewer than hematologic malignancies, but display potentials that CAR-NK cells congregate in these tumors [50].Hematologic malignancy targets.Clinical studies have demonstrated the significant efficacy of anti-CD19 CAR-T cell therapy in tumor immunotherapy [66].However, CAR-T cell therapy is constrained by various adverse effects and manufacturing challenges.CAR-NK cells present themselves as an alternative therapeutic strategy for hematological malignancies, focusing on the currently popular targets (Fig. 7).Multiple myeloma (MM) is a hematologic malignancy, and numerous CAR-T and CAR-NK cell therapies are being currently developed [125], with B-cell maturation antigen being the most popular cell therapy target [126].Furthermore, CD38 and CD138 are also common targets for MM treatment with CAR constructs [127].CD19, CD20, and CD22 are commonly employed targets for CAR-T cell therapy in B-cell lymphoma and leukemia.Two major challenges in constructing CARs against AML include shared antigen expression and heterogeneity with hematopoietic progenitor cells.Shared antigenic expression of CD123 and CD33 can result in on-target nontumor toxicity [128,129].In several cases, targeting various AML-associated antigens (such as FLT3 [130], CD123 [131], CD33 [132], CLL-1 [133], and GRP78 [134]) using multiple CARs may be necessary because certain tumor-associated antigens (TAAs) may not be expressed on all leukemia cells.

Challenges of NK cell therapy
At present, CAR-NK cells demonstrate obvious advantages compared with CAR-T cells, as summarized in previous literature [135][136][137] (Table 1).Registered clinical trials using these cells have seen a rapid increase, as shown in Table 2. Unlike CAR-T cell clinical applications, NK cells exhibit relatively acceptable toxicity adverse effects, avoiding effects such as cytokine release syndrome.Several clinical trial evidences support this, for instance, Tang et al. reported the first-in-man CD33 + CAR-NK-92 cell clinical trial, testing safety in patients with AML experiencing relapsed and refractory conditions.The dose of 5 × 10 9 cells in each patient led to no evident adverse effects [138].Additionally, Liu et al. reported phase I and II trial outcomes using CD19 CAR-NK cells, revealing a rapid response in patients with CD19 + cancer with relapsed or refractory conditions, without apparent associations between effectors and adverse effects, including neurotoxicity, cytokine release syndrome, or GVHD [66].However, challenges persist in this type of immunotherapy, summarized as follows: Low persistence.A major drawback is the lack of in vivo persistence of infused cells in the absence of cytokine support, limiting the effectiveness of NK cell immunotherapy.While exogenous cytokines have been reported to increase proliferation and persistence of adoptive NK cells [139], they can also lead to undesired adverse effects, including the expansion of suppressive immune subsets, such as Tregs [140].Rejection of allogeneic NK cells by host T cells is also a critical consideration in cell therapy using allogeneic NK cells.Further exploration of the role of transmembrane-bound IL-15 in promoting NK cell persistence is warranted.
Transport to the desired tumor site.The efficient homing of NK cells to tumor sites has been debated, as rapid homing to the tumor bed is critical for adoptive cell therapy efficacy.This process is regulated via complex interactions between NK cells and chemokines released by tumor cells [141].Various engineering approaches have been explored to enhance NK cell migration to tumor sites.For instance, NK cells have been subjected to electroporation with mRNA encoding the chemokine receptor CCR7 to enhance migration to lymph nodes expressing the chemokine CCL19 [20,142].To improve the success rate of NK cell immunotherapy in patients with solid tumors, mouse models have been used to explore novel techniques promoting NK cell translocation to tumor sites [143]; however, the effectiveness of these approaches requires further verification in clinical trials.
Immunosuppressive tumor microenvironment (TME).The TME, encompassing immunosuppressive molecules, immunosuppressive cells, and an unfavorable environment hindering immune cell function, poses a major obstacle for CAR-NK cell therapy.Immunoregulatory factors such as transforming growth factor (TGF)-β and others present in the TME can impair NK cell activity [144].Researchers are investigating the development of CAR-NK cells that counteract some of these immunosuppressive effects, such as knocking out associated genes of NK cells using CRISPR/ Cas9 technology [145].Another strategy to overcome NK cell depletion [146] in the TME is to eliminate checkpoint components using genome editing to improve their function.
Low transduction efficiency of lentivirus.Lentivirus-based transduction systems represent one of the most commonly used methods for intracellular gene modification and delivery.However, the natural resistance of NK cells to lentivirus poses a challenge to efficient transduction.Various chemicals, such as protamine sulfate, are employed to enhance viral transduction [147].
Altogether, while CAR-T cell immunotherapy provides a promising approach to treating certain cancers, there are still several limitations: 1) high costs leading to unavailability; 2) long production cycles resulting in patients being unable to afford waiting; 3) poor cell quality of patient samples potentially leading to production failure; 4) CRS and neurotoxicity contributing to high treatment risks.Therefore, the general CAR holds great promise.NK cells, with their unique biological characteristics, demonstrate distinct advantages as potential "off-the-shelf" universal CAR-NK cells.As a promising alternative, different sources of NK cells (including UCB, PB, cellular lines, and iPSCs) could be utilized.Additionally, the allogeneic context without obvious toxic adverse effects presents a significant advantage, even though most CAR-NK cells are still in preclinical or early clinical trial stages.However, the short persistence of NK cells after infusion in vivo remains a major setback.Optimization and standardization of cell expansion and target gene transfection also need further definition, considering the differences between T cells and NK cells.Lastly, akin to CAR-T cells, the lack of tumor-specific targets poses a significant challenge for CAR-NK cell applications in various cancer treatments, including hematologic and solid tumors, necessitating further development for precision medicine.

DEVELOPMENT DIRECTIONS OF NK CELL THERAPY Recognition of novel target antigens
As mentioned above, identifying highly consistently expressed target tumor antigens is a critical step in CAR design.Most TAAs are also expressed by some healthy cells, potentially causing a "targeting nontumor" effect [148].Furthermore, the expression of these TAAs can vary greatly among single-cell clones of the same tumor.To address this issue, bispecific CARs have been designed to target multiple antigens simultaneously.This can be achieved by injecting different CAR-NK cells targeting distinct antigens simultaneously or designing one CAR to recognize multiple antigens through "tandem CARs," wherein two combined elements are attached to individual molecules to enhance the immune synapse [149].Additionally, multiple CARs can be simultaneously produced on the same immune cell using a vector.

Improving NK cell activity
Various immune checkpoints, such as PD-L1, regulate and suppress NK cell activity.For instance, a new NK-92 cell line     designed with a CAR targeting PD-L1, known as PD-L1-targeting haNK, demonstrated specific antitumor effects against several tumors in preclinical data [150].Another strategy to improve the activity of CAR-NK cells involves regulating tumor metabolism, an area that has not received sufficient attention.Under hypoxic conditions, adenosine is produced via ATP metabolism by CD39 and CD73, contributing to immune evasion, preventing NK cell trafficking to tumor sites, and inhibiting NK cell maturation.NKG2D-engineered CAR-NK cells exhibited efficacy in treating lung cancer following anti-CD73 antibody inhibition [151].Therefore, immune checkpoint regulation remains a crucial consideration in cell-based immunotherapy.
Overcoming the immunosuppressive TME Tumors harbor various immunosuppressive factors, including TGFβ, IL-10, and PD-1.Several strategies are utilized to mitigate their inhibitory effects.Combining TGF-β kinase inhibitors with NK cells has been observed to restore NK cell cytotoxicity and preserve NKG2D and CD16 expression [152].Additionally, hybrid CARs incorporating extracellular TGF-β receptor domains have proven successful in improving the antitumor potential of NK-92 cells [153].Furthermore, the concurrent use of immune checkpoint blockade inhibitors presents a promising avenue.

Improving security
Enhancing the safety of CAR-NK cell-based therapy may involve modifying the CAR structure by incorporating suicide genes [154].Developing bispecific CAR molecules to better target tumorspecific antigens is another crucial approach.CAR-NK cells exhibit the unique ability to target tumors in both a CAR-dependent and CAR-independent manner.This ability can be harnessed to achieve enhanced tumor inhibition by developing nonsignaling CARs.These nonsignaling CARs lack direct killing signals but can augment the specific killing of NK cells by promoting residence and adhesion to target cells [136].Another intriguing strategy involves designing CAR-NKs capable of modulating the TME.These highly specialized CAR-NK cells express several foreign genes that can modulate the local TME to prevent any harmful effects.

Improving accessibility
Addressing the accessibility of CAR-NK cells in solid tumors necessitates various approaches, including topical, intraperitoneal, and focused ultrasound-guided drug delivery.For instance, pleural injections proved highly effective in an orthotopic model mimicking human pleural malignancies, demonstrating an even longer duration of function compared to intravenous injections [155].The topical administration of CAR immune cells may also help reduce treatment doses.

PROSPECTIVE FUTURE
NK cells stand as a unique cohort of antitumor effector cells, wielding functions such as MHC-independent cytotoxicity, cytokine production, and immune memory.These attributes position them as pivotal contributors to both innate and adaptive immune response systems.The field of CAR-NK cell therapy holds promise in clinical research, demonstrating commendable safety and preliminary efficacy in certain patients with cancer.In comparison to CAR-T cells, CAR-NK cells boast distinct advantages, yet they grapple with challenges.Enhancing cell proliferation, facilitating more efficient activation of cytotoxicity, and ultimately optimizing NK cell reconstitution are concerns.Consequently, advancements in large-scale preparation methods, cryopreservation measures, and efficacy are imperative.Addressing the short duration of in vivo persistence and exhaustion remains an unresolved frontier.Overall, CAR-NK is poised to evolve into a versatile cell product, holding greater advantages in single-drug or combined transplantation, monoclonal antibody applications, and other treatments.With the formidable antitumor lineage of NK cells as a foundation, overcoming these challenges is likely to usher in groundbreaking developments in tumor treatment.The rapid evolution of NK cell-based immunotherapy (Fig. 8), reflected in the expanding cancer cell therapy pipelines [156,157], proves that CAR-NK modifications will pave the way for new breakthroughs.In the near future, the maturation of CAR-NK cell therapy technology promises uplifting news for a broader spectrum of patients with cancer, propelling humanity closer to conquering the challenges of refractory and recurrent cancer treatments.

Fig. 2
Fig. 2 Categories of cell surface proteins expressed on NK cells.NK cells primarily express different cell surface proteins exhibiting various effects.

Fig. 3
Fig. 3 NK cells exhibit anticancer effects through diverse mechanisms.NK cells exhibit broad-spectrum anticancer effects via various mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC) via CD16 binding, secretion of cytokines (IFN-γ, TNFα), release of cytolytic granules (e.g., granzyme B), and expression of death ligands.The balance of NK cell activity is regulated by activating and inhibitory receptors.

Fig.
Fig.4Approaches in NK cell engineering against cancer.1) CAR-NK cells: NK cells that are modified to express extracellular receptors (such as scFvs, nanobodies, peptides, and ligands) for HLA-independent antigen recognition.2) TCR NK cells: NK cells that are modified to express TCR complexes for MHC-I presented cancer antigens.3) Multi-specific Ab-based NK cell therapy: NK cells that utilize mono-antibodies, bispecific antibodies, or tri-specific antibodies to recognize cancer antigens (including but not limited to bi-specific NK-cell-engager, BiKE).

Fig. 5
Fig. 5 Four primary sources of NK cells in NK cell engineering.There are four primary sources of NK cells including peripheral blood (PB), induced pluripotent stem cells (iPSC), mesenchymal stem cells (e.g., umbilical cord blood), and NK cell lines (e.g., NK-92), especially in CAR-NK cell therapy.

Fig. 6
Fig.6Generations of CARs and logic-gated control of CAR cells.A The first generation of CAR contained only CD3ζ.The second and third generations added one or two co-stimulatory domains, respectively, building on the first generation.The fourth generation introduced a cytokine secretion segment based on the third generation, and the fifth generation incorporated special binding motifs.B Logic-gated control of CAR cells was developed for precision therapy and to avoid potential toxicity, including "AND," "AND NOT," and "OR" in three logic-gated control manners.

Fig. 7 Table 2 . 2 A
Fig. 7 Popular targets in hematologic malignancy cell therapy.The currently popular targets in hematologic malignancy cell therapy are listed in the categories.

Fig. 8
Fig. 8 Updated pipelines in cancer cell therapy.Clinical pipeline changes and the year-on-year growth rate of cell therapy in the past 3 years.NK natural killer, TCR T cell receptor, TIL tumor-infiltrating lymphocyte.

Table 1 .
CAR-NK cells exhibit obvious advantages compared with CAR-T cells.